CN103614387A - Optimized porcine circovirus type-2 Cap protein gene as well as recombinant plasmid application thereof - Google Patents
Optimized porcine circovirus type-2 Cap protein gene as well as recombinant plasmid application thereof Download PDFInfo
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Abstract
本发明公开了优化的猪圆环病毒2型Cap蛋白基因及其重组质粒和应用。优化的编码PCV2Cap蛋白的基因，序列如SEQ ID NO.1、SEQ ID NO.2或SEQ ID NO.3所示。所述的重组质粒，为含有本发明所述基因的pVAX1载体。Western Blot试验结果证明这些重组在体外能够有效进行表达，并具有较好的免疫原性。动物免疫实验表明Cap蛋白改造后的重组质粒DNA能够有效诱导小鼠和猪体产生细胞和体液免疫应答，为PCV2基因疫苗的研究方面具有良好的应用前景。
The invention discloses an optimized porcine circovirus type 2 Cap protein gene, its recombinant plasmid and application. The optimized gene encoding PCV2Cap protein has a sequence as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3. The recombinant plasmid is a pVAX1 vector containing the gene of the present invention. The results of Western Blot test proved that these recombinants can be effectively expressed in vitro and have good immunogenicity. Animal immunization experiments show that the recombinant plasmid DNA modified by Cap protein can effectively induce cellular and humoral immune responses in mice and pigs, and has a good application prospect in the research of PCV2 gene vaccine.
Description
Technical field
The invention belongs to biology field, relate to carrying Cap gene of porcine circovirus type 2 gene and recombinant plasmid and the application of optimization.
Background technology
Although apply natural PCV2ORF2 constructed dna vaccine, have certain feasibility, natural ORF2 is very low at the expression amount of eukaryotic expression system, has had a strong impact on the immune effect of DNA vaccination.Studies have reported that, change the inclined to one side preferendum of PCV2ORF2 codon into Mammals pig genus and can obviously improve its expression amount in eukaryotic system.This research is passed through to modify PCV2ORF2 gene, and adopts KOZAK sequence and FMDV2A gene protein to design new Cap sequence, has further improved the effect of DNA gene vaccine.
Nucleic acid vaccine has possessed many advantages in the anti-system of epidemic disease, but it is lower to use usual way immunity to enter the efficiency of body cell, Salmonellas belongs to enterobacteriaceae, aggressive pathogenic bacterium in one group of born of the same parents, therefore effective submission antigen, excite specific humoral immunity reaction and the cell immune response of anti-salmonella and induction foreign protein, and can induce mucosa-immune and general immunity simultaneously.In recent years, by attenuation salmonella, transport DNA vaccination, be considered to a kind of up-and-coming method.
Molecule clone technology, the synthetic and pcr amplification of gene obtains gene clone after optimization process to pVAX1 carrier, and construction recombination plasmid is identified expression of recombinant proteins situation by transfection, western blot.
Animal immune experiment; to measure antibody and lymphopoiesis index after recombinant plasmid immune mouse; filter out best modifying gene; best modifying gene is proceeded to attenuation salmonella and carry out pig body immunization experiment and attack malicious Protection, prove that improved gene has good immunogenicity and immune protective.
Summary of the invention
The object of the invention is proof and take gene vaccine that improved Cap protein gene is fundamental construction immunological characteristic in animal body.
Another object of the present invention is that can preliminary identification nucleic acid vaccine for the prevention and control of PCV2.
Object of the present invention can be achieved through the following technical solutions:
The gene of the coding Porcine circovirus type 2 Cap of optimizing, sequence is as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.Wherein SEQ ID NO.1 is according to mammalian metapneumovirus preferendum, and natural PCV2ORF2 gene codon is optimized, and makes the gene that pig is had a liking for partially into, and adds KOZAK sequence, called after SynCap; SEQ ID NO.2 for being mutated into first base C after PCV2ORF2 gene start codon ATG G, called after SynCap(m on the basis of SEQ ID NO.1); SEQ ID NO.3 is for connecting two SynCap(m by 2A albumen coded sequence) gene gained, called after SynCap-2A-SynCap.
The gene of the coding Porcine circovirus type 2 Cap of described optimization, preferred sequence is as shown in SEQ ID NO.2.
The recombinant plasmid of the gene of the coding Porcine circovirus type 2 Cap that contains described optimization.
Described recombinant plasmid, is preferably the pVAX1 carrier that contains gene of the present invention, further preferably contains SynCap or SynCap(m) the pVAX1 carrier of gene.
The attenuation salmonella that contains recombinant plasmid of the present invention, preferably contains pVAX-SynCap(m) attenuation salmonella.
The application of gene of the present invention in preparing resisting porcine circovirus 2 type medicines or vaccine.
The application of recombinant plasmid of the present invention in preparing resisting porcine circovirus 2 type medicines or vaccine.
The application of attenuation salmonella of the present invention in preparing resisting porcine circovirus 2 type medicines or vaccine.
Beneficial effect
The present invention is optimized processing according to Porcine circovirus type 2 Cap sequence according to the inclined to one side preferendum of pig source pin, and has done on this basis a series of transformation and optimization.Utilize eukaryotic expression vector pVAX1 to build restructuring eucaryon plasmid pVAX-SynCap, pVAX-SynCap(m) and pVAX-SynCap-2A-SynCap.Cap albumen after the optimization of Western Blot experiment results proved can be expressed in vitro.The recombinant plasmid that mouse and pig body immunization experiment proof build and the attenuation salmonella that carries recombinant plasmid can be induced the immunne response of body effectively, PCV2 are attacked to poison simultaneously and have good immanoprotection action.Prove that constructed PCV-II nucleic acid vaccine has certain application and exploitation is worth.
The present invention builds restructuring eucaryon plasmid pVAX-SynCap, pVAX-SynCap(m) and pVAX-SynCap-2A-SynCap, these are binned in external can effectively expression Western Blot experiment results proved, and have good immunogenicity.6 weeks pVAX-SynCap(m after head exempts from) and pVAX-SynCap group ELISA antibody can be detected, 9 weeks pVAX-SynCap(m after head exempts from) group ELISA antibody reaches 1:4000, it is 1:1500 that pVAX-SynCap organizes antibody horizontal.Head exempts from latter 9 weeks, in the cell conditioned medium in each group stimulation hole, all cytokine secretion can be detected, wherein, pVAX-SynCap(m) immune group produces IL-4 and the IFN-gamma cells factor are replied higher than other each immune group (P<0.05), secondly be pVAX-SynCap and pVAX-SynCap-2A-SynCap immune group, in pVAX-Cap immune group, the secretory volume of IL-4 and IFN-γ is lower, negative control group and pVAX1 immune group minimum.
Based on above achievement in research, the visible improved recombinant plasmid dna of Cap albumen is inducing mouse and pig body generation cell and humoral immunoresponse(HI) effectively, for the research aspect of PCV2 gene vaccine has a good application prospect.
Accompanying drawing explanation
Fig. 1 Cap albumen modification scheme
SynCap:Cap albumen complete genome sequence is transformed by the inclined to one side preferendum of pig source pin;
SynCap(m): the full gene of Cap albumen of the base of suddenling change is synthetic;
SynCap-2A-SynCap: two synthetic Cap protein genes are connected by foot and mouth disease 2A gene;
The recombinate double digestion of eucaryon plasmid of Fig. 2 is identified
M:DNA Ladder DL5000; The Xho I of 1:pVAX1 and the double digestion of BamH I; The Xho I of 2:pVAX-Cap and the double digestion of BamH I; The Xho I of 3:pVAX-SynCap and the double digestion of BamH I; Xho I 4:pVAX-SynCap(m) and the double digestion of BamH I; The Xho I of 5:pVAX-SynCap-2A-Cap and the double digestion of BamH I.
The Western-blot that Fig. 3 recombinates after eucaryon plasmid transfection identifies
1：pVAX-SynCap；2：pVAX-SynCap（m）；3：pVAX-Cap；4：pVAX-SynCap-2A-Cap；5：pVAX1.
Humoral immunoresponse(HI) (a) ELISA antibody response after Fig. 4 recombinant plasmid immune mouse; (b) Neutralizing antibody response
In different time points, collect serum sample (n=5) and application indirect ELISA detection serum antibody (a) and neutralizing antibody (b).
The inverse of the titre application serum Average dilution of neutralizing antibody represents, data represent with mean value ± S.D.
Fig. 5 cellullar immunologic response after eucaryon plasmid immune mouse of recombinating
Head exempts to collect spleen cell sample (n=5) in 42 days and 63 days, and then the PCV2SH strain of application of purified stimulates, and sets 3 repetitions for every group.Stimulate after 72 hours, apply traditional mtt assay mensuration propagation and reply.Data represent with mean value ± S.D.
Fig. 6 cytokine response after eucaryon plasmid immune mouse of recombinating
Head exempts to collect spleen cell sample (n=5) in 42 days and 63 days, and then the PCV2SH strain of application of purified stimulates, and sets 3 repetitions for every group.Stimulate after 72 hours, collecting cell supernatant is measured the content of cytokine in supernatant.Data represent with mean value ± S.D.
Fig. 7 carries the recombinant plasmid immune swine of Salmonellas and attacks the rear viremia state of poison
The detection of viral level in Fig. 8 Lymphoid tissue
Fig. 9 histopathology changing conditions
Attack poison 28 days after, animal is slaughtered, fixing organization is prepared histopathologic slide, SynCap(m) (a), pVAX-SynCap(m) (b), pVAX1(c), SL3263(d) and positive control (e) (HE staining, 400 * .)
Biomaterial preservation information
PCV2SH strain, Classification And Nomenclature is porcine circovirus 2 type, on March 4th, 2008, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, be called for short CGMCC, preservation address is Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and preserving number is CGMCC No.2389.
Embodiment:
The engineered design of implementation column 1Cap gene, amplification and acquisition
In order to improve Cap protein immunization characteristic, this research is modified PCV2ORF2 gene, comprise that (1) is according to the inclined to one side preferendum of Mammals codon, natural Cap gene codon is optimized, make the gene that Mammals pig is had a liking for partially into, and add KOZAK sequence, and called after SynCap, sequence is as shown in SEQ ID NO.1; (2) on the basis of prioritization scheme (1), first base C after PCV2ORF2 gene start codon ATG is mutated into G, called after SynCap(m), sequence is as shown in SEQ ID NO.2; (3) by 2A albumen, connect two SynCap(m) gene, called after SynCap-2A-SynCap, sequence is as shown in SEQ ID NO.3.SynCap gene obtains by chemosynthesis.Synthetic gene order two ends are with BamH I and Xho I restriction enzyme site.
According to the synthetic a pair of Auele Specific Primer of PCV2ORF2 gene order design, the pMD18T-Cap that this laboratory builds of take is template, amplification Cap gene, and sequence is as shown in SEQ ID NO.4.
Upstream primer PCV2-Cap.15 '-ATTA
gGATCCgCCACCATGACGT-3 ' (SEQ ID NO.5)
Downstream primer PCV2-Cap.25 '-GCGCTCGAGTCACTTAGGGTTAAGTG-3 ' (SEQ ID NO.6), upstream and downstream primer contains respectively BamH I and Xho I restriction enzyme site.
Delivery plate pMD18T-Cap0.5 μ L, adds PCR mixed solution 24.5 μ L(2.5 μ L10 * buffer, 2 μ L dNTP(2.5mM), 1.5 μ L Mg
2+(25mM), two each 1 μ L(20pM of primer), 0.2 μ L Taq enzyme (2.5U), sterilizing distilled water 16.3 μ L).Reaction cycle parameter: 94 ℃ of denaturation 10min; 94 ℃ of sex change 45s; 58 ℃ of annealing 30s; 72 ℃ are extended 30s, carry out altogether 35 circulations; Then 72 ℃ are extended 10min.1% agarose gel electrophoresis is observed amplified production.
All the other derived sequence SynCap(m), SynCap-2A-SynCap be take the SynCap that synthesizes and through PCR and molecular cloning, is obtained as template.
For the primer of SynCap of increasing in Table 1.
The table 1 Different Ca p protein gene required primer that increases
Construction of recombinant plasmid after the transformation of implementation column 2Cap protein gene
The enzyme of 1.PCR product and pVAX1 is cut and is reclaimed
To reclaim Cap, SynCap, the SynCap(m of purifying), SynCap-2A-SynCap gene and pVAX1 carry out double digestion processing with restriction enzyme BamH I and Xho I respectively, endonuclease reaction condition is 37 ℃ of water-bath 4h, uses DNA glue to reclaim test kit PCR product and eucaryon plasmid pVAX1 enzyme are cut to product recovery purifying.
2. goal gene is cloned into pVAX1 carrier
By BamH I and Xho I restriction enzyme site, by Cap, SynCap, SynCap(m), SynCap-2A-SynCap gene is cloned into respectively in empty carrier pVAX1.16 ℃ of connections of spending the night, will connect product and transform DH5 α competent cell, the dull and stereotyped cultivation of LB that coating contains that penicillin of card, and the single bacterium colony of picking is cultivated, the conventional doubtful recombinant plasmid of alkaline lysis method of extracting.
3. the preparation of bacillus coli DH 5 alpha competent cell and conversion
The preparation of competence bacillus coli DH 5 alpha: get frozen DH5 α bacterial classification streak inoculation in not containing on antibiotic LB flat board, line on the substratum that contains resistance simultaneously and compare, 37 ℃ of overnight incubation.The single colony inoculation of picking next day in 3mL LB liquid nutrient medium, 37 ℃ of 200rpm jolting overnight incubation.Get 10 μ L bacterium liquid and be inoculated in new 3mL LB liquid nutrient medium, 37 ℃ of joltings are cultivated about 2h to OD600 and are reached 0.3-0.4.Under aseptic condition, culture is poured in the aseptic Eppendorf pipe of ice precooling, 4 ℃ of centrifugal 30s of 12000rpm, discard supernatant and are inverted control and do.0.1mol/L CaCl with 800 μ l ice precoolings
2the resuspended bacterial precipitation of solution, ice bath is placed 30 minutes, and 4 ℃ of centrifugal 30s of 12000rpm, abandon supernatant, are inverted control and do, the last 0.1mol/LCaCl with 100 μ L ice precoolings
2solution is resuspended bacterial precipitation lightly, and 4 ℃ save backup.
Connect the conversion of product: get connection product 10 μ L and join in the competence bacillus coli DH 5 alpha of 100 μ L, thumb flicks and mixes rear ice bath 30min; Eppendorf pipe is put to heat shock 90s in 42 ℃ of water-baths, then rapid ice bath 2min; The 800 μ L LB liquid nutrient mediums that add 37 ℃ of preheatings, 45min is cultivated in 37 ℃ of 200rpm joltings; After the centrifugal 30s of 12000rpm, discard most of supernatant, remain the resuspended bacterial precipitation of 30 μ L supernatant, coat containing in the resistant panel of 50 those penicillin of μ g/mL card, in 37 ℃ of incubators, cultivate 16-24h.
4. a small amount of of recombinant plasmid is extracted
Single bacterium colony after picking connects product and transforms on institute's that penicillin resistance flat board of the card that be coated with, is inoculated in containing in the LB substratum of 50 those penicillin of μ g/mL card of 3mL, 37 ℃ of 200rpm overnight shakings cultivations.Get 1.5mL bacterium liquid, the centrifugal 30s of 12000rpm, precipitation thalline; After supernatant discarded, add solution I (the 50mM glucose of 300 μ L4 ℃ precoolings completely; 25mM Tris-Cl, Ph8.0; 10mM EDTA) resuspended bacterium; The solution II 300 μ L(0.2M NaOH that add new preparation; 1%SDS), put upside down gently centrifuge tube for several times, to bacterium liquid, become limpid, afterwards centrifuge tube is placed on ice; Add the ice-cold solution III of 225 μ L (5M potassium acetate 60mL, glacial acetic acid 11.5mL, water 28.5mL), put upside down gently several to protein denaturation and become white agglomerate, put 3-5min on ice; 4 ℃ of centrifugal 10min of 12000rpm, move to supernatant in new centrifuge tube; Add isopyknic phenol: chloroform, put upside down and mix the standing 5min of rear room temperature, the centrifugal 5min of 12000rpm; Get supernatant to new centrifuge tube, add isopyknic Virahol, put upside down and mix, room temperature is placed 10min; The centrifugal l5min of 12000rpm, abandons supernatant, and by 70% washing with alcohol once, natural drying at room temperature, adds the distilled water of 20 μ L to dissolve; Add 0.5 μ L RNase A(10mg/mL), 37 ℃ of effect 30min(these steps can be at phenol: before chloroform extracting, complete); 1% agarose gel electrophoresis analysis.
5. the enzyme of recombinant plasmid is cut and is identified and sequential analysis
The doubtful recombinant plasmid of extracting identifies after (Fig. 2) through PCR and double digestion, by the evaluation of checking order of the Jun Yesongyou Invitrogen company of containing positive plasmid.Finally by correct positive recombinant plasmid pVAX-Cap, pVAX-SynCap, pVAX-SynCap(m) and pVAX-SynCap-2A-SynCap for follow-up test.
Vivoexpression and the evaluation of embodiment 3 recombinant plasmids
1. the purifying of plasmid
Method is with reference to Promega company plasmid a small amount of purification kit specification sheets.Concrete operations are as follows: respectively picking is containing restructuring eucaryon plasmid pVAX-Cap, pVAX-SynCap, pVAX-SynCap(m) and the mono-bacterium colony of DH5 α of pVAX-SynCap-2A-SynCap, be inoculated in containing in the LB substratum of 50 those penicillin of μ g/mL card of 3.0mL, 200rpm overnight shaking is cultivated.Get respectively 5 * 1.5mL bacterium liquid, the centrifugal 30s of 12000rpm, precipitation thalline; After supernatant discarded, add the resuspended bacterium of solution I (20 μ g/mLRNase A) of 250 μ L4 ℃ precoolings completely; Add 250 μ L solution II, repeatedly cracking is complete to put upside down gently centrifuge tube, and centrifuge tube is placed on ice to (being no more than 5min); The solution III that adds 350 μ L, covers tightly the mouth of pipe, and after pipe is inverted, gentleness is put upside down to protein denaturation and become white agglomerate, puts 3-5min on ice; 4 ℃ of centrifugal 10min of 12000rpm; Supernatant is transferred in adsorption column to the centrifugal 1min of 10000rpm; Filtrate was repeated to post once, to improve the rate of recovery; The WB solution that adds 500 μ L, the centrifugal 1min of 12000rpm; After discarding filtrate, repeated washing once; The centrifugal pillar 2min of 12000rpm, dry adsorption column; The Elution Buffer that adds 50 ℃ of preheatings of 50 μ L, room temperature is placed 3min, the centrifugal 1min of 12000rpm, wash-out plasmid DNA; With spectrophotometric determination OD260 and OD280, calculate respectively recombinate eucaryon plasmid content and purity.
DNA content (μ g/mL)=diluted sample multiple * 50 * OD260.
2. plasmid transfection BHK-21 cell
Method is with reference to TransFastTM Transfection Reagent(Promega) operational manual.Transfection is carried out on 24 porocyte plates, in transfection the day before yesterday, BHK-21 cell in peptic cell bottle, with the DMEM(containing 10% calf serum containing microbiotic) diluting cells, make its density reach 2.5 * 10
5individual cell/mL, on 24 porocyte plates, every hole inoculation 1.0mL, puts 5%CO
2, cultivate under 37 ℃ of saturated humidities; 4h before transfection, is replaced by new nutritive medium the nutritive medium of 24 porocyte plates; Take out the transfection reagent of-20 ℃ of preservations, room temperature is melted and vortex; In the 1.5mL of four sterilizings centrifuge tube, first add 200 μ L not contain the DMEM of serum, then the restructuring eucaryon plasmid pVAX-Cap, pVAX-SynCap, the pVAX-SynCap(m that add respectively 0.5 μ g) and pVAX-SynCap-2A-SynCap, after mixing, add the TransFastTM Transfection Reagent(of 3.0 μ L to make lipid: volume/μ L of DNA is 2: 1 with quality/μ g ratio) after vortex immediately, at room temperature incubation TransFastTM Transfection Reagent/DNA mixture l0-15min; From carbon dioxide cell incubator, take out cell plate, supernatant discarded; Of short duration vortex TransFastTM Transfection Reagent/DNA mixture, joins cell surface gently mixture again; Mix gently, make liposome/DNA mixture cover cell face, then immediately cell plate are put into CO2gas incubator.After incubation 1.0h, add gently the DMEM(of 2% calf serum of 1.0mL37 ℃ of preheating not contain microbiotic), cell is put back to CO2gas incubator, cultivate 3d for 37 ℃.The control wells of transfection pVAX1 and untransfected is set.
3.Western blot identifies the expression of albumen
3.1SDS-PAGE
Cell plate after transfection are kept to-20 ℃, and multigelation 3 times, cell collected.Get appropriate enchylema and add 1/4 volume 5 * electrophoresis sample-loading buffer (0.1M Tris-HCl(pH6.18), 0.2M DTT, 4%SDS, 0.12% bromjophenol blue, 20% glycerine), 100 ℃ are boiled 5min, instantaneous centrifugal after, carry out 12% polyacrylamide sepharose (SDS-PAGE) electrophoresis.
3.2Western?blot
SDS-PAGE gel, NC film and Whatman filter paper are put in transfer printing damping fluid and soaked 10min; Transfer printing is sequentially :+the utmost point-sponge-NC film-gel-sponge--the utmost point, and volume increases successively, and half-dried transfer printing condition is 20V, 30min; Transfer printing is complete, and the ponceau dyeing NC film with 5%, to determine transfer printing effect; Use PBST rinsing, wash-out ponceau.
NC film is placed in to 10% skimming milk (PBST dilution), 37 ℃ are shaken and hatch 2h; With PBST washing 3 times, each 5min; The primary antibodie (PCV25H7 monoclonal antibody, 1:10000 dilution) that adds PBST dilution, 37 ℃ are shaken and hatch 2h; With PBST washing 3 times, each 5min; Sheep anti-mouse igg-HRP(1:15000 with PBST dilution), 37 ℃ are shaken and hatch 45min; With PBST washing 5 times, each 5min; Dry NC film, equivalent mixes A liquid and the B liquid of appropriate chemical luminous substrate ECL, and mixed solution is directly added drop-wise on NC film, hatches 2-5min; With preservative film, wrap, in darkroom, on x-ray film, expose, after development, photographic fixing, observe, the results are shown in Figure 3.Western Blot experiment results proved pVAX-SynCap, pVAX-SynCap(m), pVAX-SynCap-2A-Cap recombinant plasmid all can effectively express in vitro, and have good immunogenicity.
1. a large amount of preparations of plasmid
Method is with reference to embodiment 3.10 μ L bacterial classifications are inoculated in to containing in the LB substratum of 50 those penicillin of μ g/mL card of 3mL, 37 ℃ of 200rpm shaking culture 6h; 3mL bacterium liquid is all poured into containing in the 200mL LB substratum of 50 those penicillin of μ g/mL card, and 37 ℃ of 200rpm overnight shakings are cultivated; Collect the centrifugal 10min of bacterium liquid 7500rpm next day, after supernatant discarded, add STE(10mM Tris-HCl, 0.1M NaCl, 1mM EDTA, the pH8.0 of 100mL4 ℃ of precooling completely), resuspended precipitation; The centrifugal 10min of 7500rpm; Solution I (the 50mM glucose that adds 2mL4 ℃ of precooling after supernatant discarded; 25mM Tris-Cl, PH8.0; 10mM EDTA) abundant resuspended bacterium, and minute be filled in EP pipe 200 μ L/ pipes; The solution II 400 μ L(0.2M NaOH that add new preparation; 1%SDS), put upside down gently centrifuge tube for several times, centrifuge tube is placed on ice, to bacterium liquid, substantially become bright; Add the ice-cold solution III of 300 μ L (5M potassium acetate 60mL, glacial acetic acid 11.5mL, water 28.5mL), put upside down fast several to protein denaturation and become white agglomerate, put 3-5min on ice; 4 ℃ of centrifugal 10min of 12000rpm, move to supernatant in new centrifuge tube; The Virahol that adds 0.6 times of volume, mixes and is placed on-20 ℃ more than 1h; 4 ℃ of centrifugal 10min of 12000rpm; Abandon supernatant, precipitation by 70% washing with alcohol once; 4 ℃ of centrifugal 5min of 12000rpm, add the distilled water of 50 μ L to dissolve after natural drying at room temperature; Add 5 μ L RNase A(10mg/mL), after 37 ℃ of effect 30min, be incorporated into a pipe; Add isopyknic phenol: chloroform, vortex mixes rearmounted room temperature 5min, 4 ℃ of centrifugal 5min of 12000rpm; Get supernatant to new centrifuge tube, add isopyknic chloroform, put upside down and mix, 4 ℃ of centrifugal 5min of 12000rpm; Get supernatant to new centrifuge tube, add 40 μ L NaAc(pH5.2) and 0.9mL dehydrated alcohol, put upside down and mix, be placed in-20 ℃ more than 1h; 4 ℃ of centrifugal 10min of 12000rpm, abandon supernatant, by 70% washing with alcohol once; 4 ℃ of centrifugal 5min of 12000rpm, add the distilled water dissolution precipitation of 1mL ,-20 ℃ of preservations after natural drying at room temperature; With spectrophotometric determination OD260 and OD280, calculate respectively recombinate eucaryon plasmid content and purity.
DNA content (μ g/mL)=diluted sample multiple * 50 * OD260.
2. mouse immuning test
2.1 mice group and immunization experiment
Get 90 of female Balb/c mouse in age in 6-8 week, be divided into 6 groups, 15 every group.First, second, third and fourth, five groups of respectively leg muscle injecting immune pVAX-SynCap, pVAX-SynCap(m), pVAX-Cap, pVAX-SynCap-2A-SynCap and pVAX1, dosage is every mouse immune 100 μ g, the 6th group of negative control group, injection PBS, 21, after 42 days respectively with identical dosage booster immunization (24h before immune plasmid, leg muscle is injected 2% Xylotox, and 50 μ L/ only).Respectively at after first immunisation 42 and 63d blood sampling measure ELISA antibody and neutralizing antibody; Get spleen, separated lymphocyte, measures lymphproliferation response; And measure cytokine response after spleen lymphocyte stimulated in vitro.
2.2ELISA antibody test
2.2.1Cap-C the preparation of albumen
(1) design of primers
According to PCV2SH pnca gene sequence (AY686763) design primer, amplification ORF251-234 fragment, and called after Cap-C.Upstream and downstream primer is respectively introduced BamH I and Xho I site.Sequence is as follows:
Cap51F5’-AA
GGATCCCGCACCATCGGTTATAC-3’(SEQ?ID?NO.12)
Cap234R5’-CTT
CTCGAGTCACTTAGGGTTAAGTGGG-3’(SEQ?ID?NO.13)
Above primer is synthetic by Shanghai Invitrogen company.
(2) pcr amplification
Adopt conventional phenol chloroform extraction method to extract viral PCV-SH (CGMCC No.2389) strain DNA: get virus liquid 500 μ L, adding final concentration is SDS and the Proteinase K that is respectively 1% and 50 μ g/ μ L, 55 ℃ of effect 30min; Add isopyknic phenol extracting, vibration mixes, the centrifugal 10min of 12000rpm; Get supernatant liquor, add the extracting of isopyknic phenol/chloroform, vibration mixes, the centrifugal 10min of 12000rpm; Get supernatant liquor, add isopyknic chloroform extracting, the centrifugal 10min of 12000rpm; Get supernatant liquor, add the 3M sodium acetate (pH5.2) of 1/10 volume and the dehydrated alcohol of 2.5 times of volumes ,-20 ℃ are spent the night, precipitation DNA; Then the centrifugal 15min of 12000rpm, discards supernatant liquor, drying at room temperature; Finally add the aseptic ddH of 20 μ l
2o dissolves.Take it as template, carry out pcr amplification.PCR reaction system is: upstream, each 1.0 μ L of downstream primer (10pmol/L); MgCl
2(25mmol/L) 1.5 μ L; DNTP s(2.5mmol/L) 2.0 μ L; 10 * PCR buffer2.5 μ L; The plasmid template 2.0 μ L of dilution; Taq enzyme (5U/ μ L) 0.2 μ L; Sterilizing distilled water is mended to 25 μ L.Loop parameter: 95 ℃ of denaturation 5min; With 94 ℃ of 45s, 54 ℃ of 45s, 72 ℃ of 45s carry out 35 circulations; Last 72 ℃ are extended 10min.1% agarose gel electrophoresis is identified PCR product.Through 1% agarose gel electrophoresis analysis, result is presented at corresponding position all a specific band, conforms to the big or small result of expection
(3) structure of recombinant plasmid and enzyme are cut evaluation
Pcr amplification object simultaneously, is extracted pET32a(+ according to plasmid extraction kit specification sheets) plasmid.After electrophoresis finishes, according to test kit specification sheets, reclaim PCR product, PCR product and plasmid reclaim purifying again through BamH I/Xho I double digestion respectively.
Endonuclease reaction system is as follows:
The PCR product that the enzyme that recovery is obtained is cut and carrier pET-32a(+) 16 ℃ spend the night and be connected.
Linked system is as follows:
Then transform DH5 α competent escherichia coli cell, the LB that coating contains penbritin is dull and stereotyped, cultivates 18h left and right for 37 ℃.The some single bacterium colonies of picking, 37 ℃ of inoculation LB liquid nutrient mediums are cultivated 16-18h, and conventional alkaline lysis method of extracting plasmid is cut and is identified that positive plasmid send the evaluation of checking order of Invitrogen company through PCR, enzyme.Identify that accurate positive plasmid pET-32a-CapC is for follow-up test.
(4) expression of Cap-C albumen
Through the correct recombinant plasmid transformed BL21 of sequencing, simultaneously by empty plasmid pET-32a with method transformed competence colibacillus e. coli bl21.Cultivate 24h for 37 ℃, picking list colony inoculation is in the LB liquid nutrient medium that contains penbritin, and 37 ℃ of joltings of spending the night are cultivated.
1. the bacterium liquid 10 μ L that get incubated overnight are inoculated in the LB liquid nutrient medium that 3ml contains penbritin (50 μ g/mL), and 37 ℃ of 200rpm shaking culture 3h left and right, make OD
600reach 0.6-0.8, get 100 μ L samples and as induction is front, contrast in aseptic Eppendorf pipe;
2. to adding final concentration in above-mentioned bacterium liquid, be the abduction delivering that the IPTG of 1.0mmol/L carries out recombinant protein, and after adding IPTG the 2nd, 3,4,5,6h sampling;
3. 4 ℃ of bacteriums that the centrifugal 5min of 8000rpm collects abduction delivering, PBS(pH7.2) resuspended, so washs 2 times;
4. abandon supernatant completely, with the resuspended bacterial precipitation of appropriate PBS;
5. by bacterium liquid multigelation 3 times;
6. ultrasonic treatment bacterium: power is selected 200W operates on ice chest, ultrasonic degradation 5s, and pause 5s, until bacterium liquid becomes limpid;
7. 4 ℃ of centrifugal 10min of 8000rpm, collect respectively supernatant and precipitation: precipitation is used with the isopyknic PBS of supernatant resuspended, it is standby that supernatant and precipitation suspension are respectively got 100 μ L.
8. in sample, add 5 * protein sample sample-loading buffer, fully mix latter 100 ℃ and boil 5min, centrifugal before loading, draw supernatant and carry out SDS-PAGE.
9. the evaluation of Cap-C albumen
PET-32a-Cap-C is transformed to BL21, and picking recombinant bacterium carries out abduction delivering, analyzes discovery recombinant bacterium all occur an obvious protein band through SDS-PAGE after induction 4h, consistent with expection, shows that Cap-C recombination fusion protein obtains better expression.Thalline is carried out to ultrasonic degradation, and in separation, cleer and peaceful inclusion body precipitation, finds that target protein is mainly present in inclusion body.Target protein is carried out to purifying, and by the dilution method combining of dialysing, target protein is carried out to renaturation, target protein obtains good purifying.By Western blot, further the antigenicity of recombinant protein is identified, result demonstration recombinant C ap-C albumen can be identified by Histidine specific monoclonal antibody, show that recombinant protein is with the fusion protein form expression with histidine-tagged, and there is good antigenicity.
2.2.2ELISA antibody test
Recombinant C ap-C albumen 2 μ g/mL coated elisa plates with purifying, proceed to 4 ℃ after 37 ℃ of 2h and spend the night; PBST washing 3 times, each 5min; 5% skimming milk, 200 μ L/ holes, 37 ℃ of sealing 2h; PBST washing 3 times; Serum to be checked is made to doubling dilution, each dilution serum sample is added to enzyme mark hole, each sample repeats 2 holes, and same method is set up the negative mouse serum contrast of PCV2 simultaneously, and sets positive control, 100 μ L/ holes, 37 ℃ of effect 1h; ; PBST washing 3 times; By after goat anti-mouse igg-HRP1:20000 dilution, add enzyme mark hole, 100 μ L/ holes, 37 ℃ of effect 0.5h; PBST washing 3 times; Add equal-volume to mix A liquid and the B liquid of TMB nitrite ion, 100 μ L/ holes, 37 ℃ of effect 15min; The H that adds 2M
2sO
4termination reaction, 50 μ L/ holes; Measure the value of OD450nm.Meet following condition and be judged to the positive, otherwise negative, to using and judge that positive serum greatest dilution is as the antibody titers of this sample, the ELISA antibody that can find out pVAX-SynCap (m) induction from Fig. 4 a is higher than other group.Exempt from after 6 weeks pVAX-SynCap(m) and pVAX-SynCap group ELISA antibody can be detected, 9 weeks pVAX-SynCap(m after head exempts from) group ELISA antibody reaches higher level (about 1:4000), pVAX-SynCap group antibody horizontal is 1:1500, the antibody horizontal of the two is all significantly higher than other group (P<0.01), and pVAX1, pVAX-Cap and pVAX-SynCap-2A-SynCap and negative group all do not detect PCV2 specific antibody.
Sample/Negative（S/N）＝ODsample/ODnegative≥2.1。
2.3 virus neutralization tests
By PCV2SH strain virus freeze thawing 3 times, it is standby that the centrifugal 5min of 10000g gets supernatant.Before virus titer is measured, 24h left and right is by PK15 cell seeding 96 porocyte plates, cell covers with after individual layer, by maintenance medium (containing the DMEM of 2% calf serum), virus is made to 10 doubling dilutions, and the virus liquid having diluted to cell inoculation, each extent of dilution is done 4 repeating holes, 100 μ L/ holes; Set virus stock solution used and maintenance medium contrast simultaneously; Under 37 ℃, 5%CO2 saturated humidity, cultivate 3d, and carry out indirect immunofluorescence assay, observe fluorescence, each dilution fluorescence hole count that counts, calculates viral TCID50 by Karber method.
Before virus neutralization tests, 24h left and right is by PK15 cell seeding 96 porocyte plates, 56 ℃ of deactivation 30min of serum to be checked, after the centrifugal 5min degerming of 10000rpm, careful sucking-off supernatant, by maintenance medium (2%DMEM), serum is pressed 1: 4,1: 8,1: 16,1: 32,, make 2 continuous doubling dilution, and be inoculated on the cell that covers with individual layer; Concrete operations are as follows: get serum to be checked and negative control sera 150 μ L, after 56 ℃ of deactivation 30min, the centrifugal 5min of 10000rpm, careful sucking-off supernatant 120 μ L, join in the DMEM of 360 μ L, serum is done to dilution in 1: 4, after mixing, therefrom take out again 240 μ L and join in the maintenance medium that 240 μ L are new, do dilution in 1: 8, the like; Virus is diluted to the L containing 200TCID50/100 μ by maintenance medium, then the serum of dilution is mixed with isopyknic virus liquid (containing 200TCID50/100 μ L), in 37 ℃ of water-bath effect 1h.Discard the nutritive medium in 96 well culture plates that cover with individual layer PK15 cell, serum-virus mixed solution is joined in cell hole, 100 μ L/ holes, each serum dilution is made 4 repeating holes, cultivates 4-5d for 37 ℃.Set following contrast simultaneously: (I) virus-negative serum contrast, (II) negative serum contrast, (III) blank, and carry out indirect immunofluorescence assay.Result judges, when fluorescence appears in virus-negative serum control wells, and the fluorescence hole count of each serum dilution is recorded in negative serum and blank hole while all there is not fluorescence.The serum greatest dilution that Fluorophotometry occurs completely of usining is tired as the neutralization of serum to be checked, the results are shown in Figure 4b, can find out that the neutralizing antibody of pVAX-SynCap (m) induction is higher than other group.
2.4 lymphocyte proliferation assay
PCV2SH virus is adjusted to 25 μ g/mL as stimulator antigen with the RPMI-1640 substratum containing 10% serum, and every hole adds 100 μ L, and control wells adds 100 μ L containing the RPMI-1640 substratum of 10% serum, respectively does 3 repeating holes.At 37 ℃, cultivate 66h, then every hole adds 40 μ L MTT(5mg/mL), continue to cultivate 4h at 37 ℃.Nutrient solution is abandoned in suction, and every hole adds 100 μ L DMSO, and crystallization is melted in vibration, measures the value of OD570nm.Calculating stimulation index: stimulation index SI=stimulates the OD value in hole/do not stimulate the OD value in hole.The results are shown in Figure 5.
2.5 cytokine assay
Collect the immunity cell conditioned medium that in the time of latter 63 days, in lymphocyte proliferation assay, each group stimulates hole 72h, mix rear mensuration wherein IL-4 and the IFN-γ content ，An U.S. R & D mouse IL-4 of company and IFN-γ specification sheets carry out, the results are shown in Figure 6.Result shows: head exempts from latter 9 weeks, in the cell conditioned medium in each group stimulation hole, all cytokine secretion can be detected, wherein, pVAX-SynCap(m) immune group produces IL-4 and the IFN-gamma cells factor are replied higher than other each immune group (P<0.05), secondly be pVAX-SynCap and pVAX-SynCap-2A-SynCap immune group, in pVAX-Cap immune group, the secretory volume of IL-4 and IFN-γ is lower, negative control group and pVAX1 immune group minimum.
3. the structure of recombinant attenuated Salmonellas and evaluation
The preparation of 3.1 attenuation salmonella competent cells
Get frozen Salmonella typhimurium SL3263 streak inoculation non-resistant LB dull and stereotyped, 37 ℃ of incubated overnight; Single colony inoculation 5mL LB liquid nutrient medium on picking flat board, 37 ℃ of overnight shakings are cultivated; Every 5mL LB liquid nutrient medium is inoculated 100 μ L overnight culture, and 37 ℃, 200rpm shaking culture, to OD600nm=0.6; Culture ice bath 30min, the centrifugal 10min of 4000rpm; Abandon supernatant, with the resuspended bacterium of WB solution of equal-volume ice bath, the centrifugal 10min(WB=10% glycerine of 4000rpm, 90% distilled water, filters); Repeat previous step 2 times; Discard most of WB, making residual volume is that the every 10mL primary culture of 50 μ L(is prepared competent cell 50 μ L, 0.5%) and, mix and be competent cell.
3.2 electricity transform
In 40 μ L competent cells, add 4 μ L plasmid pVAX-SynCap(m), mix; Mixed solution adds in the cold pole cup of ice bath, ice bath 5min; Then carry out electricity and transform, parameter is set to 2500V, 200 Ω, 25 μ F[11], t=4.3s; Separately get electric the conversion in contrast of competent cell that does not add plasmid; After electric shock, in pole cup, add 500 μ L LB immediately, mix; Proceeded to rapidly after EP pipe 37 ℃ of shaking culture 40min; Get 100 μ L coatings dull and stereotyped containing the LB of 50 those penicillin resistances of μ g/mL card, 37 ℃ of incubated overnight.
3.3 identify
The single bacterium colony of the doubtful positive of picking, is inoculated in containing in the LB liquid nutrient medium of 50 those penicillin of μ g/mL card, is about 1.0 employing alkali cracking methods extracts plasmid to OD600nm, and plasmid is transformed to DH5 α competent cell again, adopt alkali cracking method again to extract plasmid, double digestion is identified, 37 ℃ of effect 2h.Enzyme cut after 1% agarose electrophoretic analysis.
4. piglet protest test
23 piglets are divided into 5 groups at random, every group of 4-5 head, isolated rearing, as Fig. 5-1.The 1st group of inoculation plasmid pVAX-SynCap(m) (500 μ g/ head), the 2nd group of oral immunity SL-pVAX-SynCap(m) (108CFU/mL), the 3rd group of immunity PBS2mL/ head, the 4th group of inoculation plasmid pVAX1(500 μ g/ head), the 5th group of oral immunity SL3263(108CFU/mL).Immunity two minor tick three weeks.Two exempt within two weeks, with PCV2SH strain, to attack poison, every virus liquid that pig collunarium 3mL TCID50 is 1.0-5.0/mL, each 1.5mL of each nostril afterwards.Respectively after first immunisation 21 days (two exempt from before), 35 days (attacking before poison) and attack poison after blood sampling in the 4th day, 7 days, 11 days, 19 days, 28 days, separation of serum, detects for PCV2 specific ELISA antibody, neutralizing antibody and viremia.Attack the poison same day and cut open every pig while killing and weigh respectively, for calculating every relative day weight gain of pig [body weight during body weight when RDWG=(cuts open and kills-attack poison)/28/ body weight while attacking poison].Attack the clinical manifestation of poison rear every day of observed and recorded pig, measure every pig rectal temperature.All groups of laboratory animal are cutd open inspection in latter 28 days in attacking poison, observe and record lymphoglandula and lungs pathological change, gather lymphoglandula and lungs sample for the preparation of tissue pathological slice.
Table 2 piglet immunological protection test design
4.1PCV2 specific ELISA antibody test and neutralizing antibody detect
The same 2.2.2 of ELISA antibody detection method, enzymic-labelled antibody changes the SPA-HRP of 1:20000 dilution into.Respectively at one, exempt to exempt from for latter 21 days, two within latter 14 days, to gather serum, measure neutralizing antibody, method is with 2.3.
Head exempts from latter 3 weeks, and pVAX-SynCap(m) immune group has produced the ELISA antibody horizontal of 1:40, and SL-pVAX-SynCap(m) immune group has produced the ELISA antibody horizontal of 1:50, and all the other each groups all do not detect ELISA antibody and neutralizing antibody.Head exempts from latter 5 weeks, pVAX-SynCap(m) immune group ELISA antibody horizontal is 1:150, neutralizing antibody level is 0, SL-pVAX-SynCap(m) immune group ELISA antibody horizontal is 1:280, neutralizing antibody level is 0, other each groups do not detect PCV2 specific antibody, illustrate that restructuring eucaryon plasmid and recombinant salmonella have certain immune efficacy, but its antibody titer causing are not high.
Table 3 test pig ELISA and neutralizing antibody measurement result
4.2 serum and Lymphoid tissue PCV2 viral level detect
4.2.1 the extraction of sample DNA
Serum DNA extraction: get 200ul serum, add 400ulPBS, the Proteinase K of the SDS of final concentration 1% and final concentration 50 μ g/mL, 56 ℃ of water-bath 30min, add isopyknic phenol, and vibration mixes, the centrifugal 10min of 12000rpm; The careful supernatant of drawing, to new EP pipe, adds isopyknic phenol/chloroform, and vibration mixes, the centrifugal 10min of 12000rpm; The careful supernatant of drawing, to new EP pipe, adds isopyknic chloroform, and vibration mixes, the centrifugal 10min of 12000rpm; The careful supernatant of drawing, to new EP pipe, adds the dehydrated alcohol of the 3M sodium acetate (pH5.2) of 1/10 volume and the precooling of 2.5 times of volumes ,-20 ℃ of precipitation DNA that spend the night; The centrifugal 15min of 12000rpm, abandons supernatant, precipitates by 75% washing with alcohol once drying at room temperature; Finally add 20 μ L aseptic double-distilled water dissolving DNAs ,-20 ℃ save backup.
Tissue DNA extracts: gets 0.2g inguinal lymph nodal tissue, shreds, add homogenate after 1.2ml PBS, and multigelation three times, the centrifugal 10min of 4000rpm, gets 50ul supernatant in new EP pipe, adds 500ul DNAZOL, and vortex mixes the standing 10min of rear room temperature; The dehydrated alcohol that adds 250ul precooling, after mixing in 4 ℃ of standing 5min; The centrifugal 10min of 12000rpm, abandons supernatant, precipitates by 75% washing with alcohol once drying at room temperature; Finally add 20 μ L aseptic double-distilled water dissolving DNAs ,-20 ℃ save backup.
4.3.2Real-time?PCR
Real-time PCR reaction system is: DNA2 μ L, 2 * Power SYBR Green PCR MasterMix(TOYOBO company) 10 μ L, each 1 μ L(final concentration 400nM of upstream and downstream primer, PCV2F:5 '-CCAGGAGGGCGTTCTGACT-3 ' (SEQ ID NO.14); PCV2R:5 '-CGTTACCGCTGGAGAAGGAA-3 ' (SEQ ID NO.15)), aseptic double-distilled water is supplied volume to 20 μ L.Put on ABI7300real time PCR instrument and carry out, response procedures is: 95 ℃ of 2min of denaturation, and 95 ℃ of 15s, 60 ℃ of 1min, carry out 40 circulations.Set negative control simultaneously and use pT-SH10 times of doubling dilution of positive plasmid template as template, by same method operation, drawing standard curve, Ct value and typical curve calculate corresponding copy number per sample, see Fig. 7 and 8.
4.4 body temperature change and lungs cardinal principle pathology statistics
After attacking poison, regularly measure the body temperature of swinery every day, and make a record.Attack poison latter 28 days, carry out according to a conventional method pathological anatomy, observe lymphoglandula, lungs pathology substantially, according to pathology weight, pathological change is divided into 3 grades.And take lungs and lymph node tissue to fix by 4% formaldehyde solution, for the preparation of follow-up pathological section.General pathology changes in Table 4.Proved that the pathological change that pVAX-SynCap (m) organizes each histoorgan will significantly be lighter than other groups.
Table 4 is respectively organized pig lungs lymphoglandula and is observed pathology and histopathology changing conditions
-indicate without pathology; + expression Minimal change; ++ represent moderate pathology; +++ represent serious pathology
4.5 pathological study
4.5.1 the preparation of histopathologic slide
From the lungs of animal and lymphoglandula, take off and be not more than the tissue block that 2cm * 4mm is thick, put into rapidly 50mL4% formaldehyde solution fixing; Tissue sample repeatedly rinses with PBS and soaks 6h, removes unnecessary fixing agent; Gradient alcohol dehydration: 75%:1h, 75%:1h, 85%:1h, 85%:1h, 95%:1h, 95%:1h, 100%:1h, 100%:1h.Transparent 10min left and right in dimethylbenzene, puts saturating wax 2-3h in 60 ℃ of paraffin, and tissue block is embedded in old paraffin the most at last.Paraffin-embedded tissue piece is whittled into suitable size and shape, is sandwiched on slicing machine, regulate slice thickness and cut into slices.
4.5.2 paraffin-embedded tissue section statining
Dimethylbenzene dewaxing 2 times, each 5-10min.The dyeing cup of preparing to fill respectively 100%, 95%, 85% and 75% ethanol, immerses each 1min, 1.5min, 2min, 2.5min successively by section; Tap water rushes 1-2min; Brazilwood extract dyeing 1min, washing oil blackeite (grasping under the microscope dye levels).Yihong dyeing 30s-1min, washing 1-2min.95%, 5min, * 3 times; 100%, 5min, * 2 times.Dimethylbenzene is transparent: 5-10min, * 2 times.After neutral gum sealing, observe, the results are shown in Figure 9.The pathology of lungs and lymphoglandula be divided into three grades (: without pathology; +: there is Minimal change; ++: moderate pathology; +++: serious pathology).In Table 4.Lesion tissue observations shows: pVAX1 immune group, SL3263 immune group and nonimmunely attack that all pigs of malicious control group all occur that obvious alveolus wall thickens, monocyte infiltration, bronchial secretion increases and the interstitial pneumonia such as intra-alveolar edema, there is Lymphocyte depletion in lymphoglandula, lymph follicle obscurity boundary or disappearance, pVAX-SynCap(m) immune group presents medium lesion degree substantially, and SL-pVAX-SynCap(m) pathology is slighter, blank group lungs and lymphoglandula normal (Fig. 9).Above result shows, SL-pVAX-SynCap(m) and pVAX-SynCap(m) can alleviate by attacking poison and infect the lungs that cause and the lesion tissue of lymphoglandula.
Note: in above-described embodiment, apply SPSS software, to data statistic analysis, carry out ANOVA and LSD multiple analysis, respectively organize difference, P<0.05 represents significant difference, and P<0.001 represents that difference is extremely remarkable.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107446028A (en) * | 2017-07-21 | 2017-12-08 | 江苏南农高科技股份有限公司 | Artificial reconstructed PCV2 Cap proteins, recombinant virus and its application |
| CN107841507A (en) * | 2017-11-23 | 2018-03-27 | 南京农业大学 | A kind of porcine circovirus 2 type Cap cell-penetrating peptides antigen-4 fusion protein gene of high efficient expression and its application |
| WO2018196836A1 (en) * | 2017-04-28 | 2018-11-01 | 普莱柯生物工程股份有限公司 | Porcine circovirus type 3 immunogenic composition, preparation method, and application |
| CN111560386A (en) * | 2020-03-13 | 2020-08-21 | 山东省农业科学院畜牧兽医研究所 | Soluble porcine circovirus type 2 Cap protein and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101401936A (en) * | 2008-01-23 | 2009-04-08 | 山东省农业科学院畜牧兽医研究所 | Method of preparing coexpression PRRSV ORF5 and PCV2 ORF2 bivalent nucleic acid vaccine |
| CN103255171A (en) * | 2013-03-07 | 2013-08-21 | 江苏省农业科学院 | Recombinant virus-like particles of porcine circovirus type 2 codon-optimized ORF2 gene |
-
2013
- 2013-11-22 CN CN201310594258.4A patent/CN103614387B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101401936A (en) * | 2008-01-23 | 2009-04-08 | 山东省农业科学院畜牧兽医研究所 | Method of preparing coexpression PRRSV ORF5 and PCV2 ORF2 bivalent nucleic acid vaccine |
| CN103255171A (en) * | 2013-03-07 | 2013-08-21 | 江苏省农业科学院 | Recombinant virus-like particles of porcine circovirus type 2 codon-optimized ORF2 gene |
Non-Patent Citations (2)
| Title |
|---|
| HUANG W.J. 等: "ABV21950", 《GENBANK》, 16 September 2007 (2007-09-16) * |
| 宋勤叶: "表达猪圆环病毒2型衣壳蛋白重组质粒的构建及免疫效应", 《中国博士学位论文全文数据库 农业科技辑》, no. 5, 15 September 2005 (2005-09-15), pages 050 - 79 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018196836A1 (en) * | 2017-04-28 | 2018-11-01 | 普莱柯生物工程股份有限公司 | Porcine circovirus type 3 immunogenic composition, preparation method, and application |
| CN107446028A (en) * | 2017-07-21 | 2017-12-08 | 江苏南农高科技股份有限公司 | Artificial reconstructed PCV2 Cap proteins, recombinant virus and its application |
| CN107841507A (en) * | 2017-11-23 | 2018-03-27 | 南京农业大学 | A kind of porcine circovirus 2 type Cap cell-penetrating peptides antigen-4 fusion protein gene of high efficient expression and its application |
| CN107841507B (en) * | 2017-11-23 | 2021-05-11 | 南京农业大学 | A highly expressed porcine circovirus type 2 Cap-penetrating peptide fusion protein gene and its application |
| CN111560386A (en) * | 2020-03-13 | 2020-08-21 | 山东省农业科学院畜牧兽医研究所 | Soluble porcine circovirus type 2 Cap protein and application thereof |
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